Method, detector, and apparatus for colorimetric detection of chemical and biological agents

ABSTRACT

A colorimetric detector for chemical and biological agents or toxins is made of a giant unilamellar vesicle (GUV) having a membrane bilayer which is polymerized to stabilize the giant unilamellar vesicle and to provide extended conjugated polymer backbone, and the GUV has at least one incorporated molecular recognition site for the chemical and biological agents or toxins. The GUVs are about 10-300 microns and preferably made of a polymerizable diacetylenic GUV where the acyl chains are crosslinked. When the agents or toxins bind to the recognition site the detector exhibits a color change. The detector can be used in a colorimetric detector apparatus where the samples can be present in air or in water.

This is a divisional application of copending application Ser. No.09/759,149 filed on Jan. 16, 2001 now U.S. Pat. No. 6,541,270. Theentire contents of application Ser. No. 09/759,149 are incorporatedherein by reference.

FIELD OF THE INVENTION

This invention relates to the use of giant unilamellar vesicles (GUV) inthe colorimetric detection of chemical and biological agents by simplereceptor-ligand interaction.

BACKGROUND OF THE INVENTION & DESCRIPTION OF RELATED ART

Diacetylenes and their analogues upon polymerization produce a deep bluecolored polydiacetylene polymer. The presence of conjugated single,double, and triple bonds in polydiacetylene backbone is responsible forthe deep color. See B. Tieke, G. Lieser, J. Colloid Interface Sci., 88,471(1982).

A blue to red color shift in polymer-backbone is observed when thepolymer goes through mechanical or thermal stress. See A. Singh, R. B.Thompson, J. M. Schnur, J. Am. Chem. Soc. 108, 2785(1986). This stressassociated color change in polydiacetylenic backbone has been used inthe development of various detector and sensor schemes.

This idea has been extended to the detection of molecular species whichcause stress on the polymer backbone upon binding to the molecular sitescovalently attached to the polydiacetylene backbone. For example,polydiacetylene films containing glycolipid sites have been used in thedetection of biological species such as cholera toxin. See Charych etal., Science, 261, p. 585-588 (Jul. 30 1993).

The conventional approach of thin polydiacetylenic film faces thedifficulty of making the thin film and then transferring it onto asubstrate. Reading the film spectrophotometrically poses an additionalproblem. The film is generally deposited on the glass slide substrateand the binding experiment is done in the solution for about 20 minutes.The color changes are read spectrophotometrically, a cumbersome processwhich may lead to some inaccuracies due to changes in the medium duringthe course of the experiment.

Vesicles serve as the best substrates because of their large surfacearea, good dispersion behavior, and availability of surface availablereceptor sites for toxins. But the diacetylene moiety in a small vesicledoes not polymerize efficiently due to its small radius of curvature.Therefore color changes due to stress on the polymer backbone,transferred by ligand binding, may not be visible.

Multilamellar vesicles (MLVs) may polymerize better than smallunilamellar vesicles (SUVs), but they will partially transform intoother structures such as tubules in most cases when cooled below theirphase transition temperature. See P. Yager, P. E. Schoen, Mol. Cryst.Liq. Cryst., 106, 371(1984). This cooling step is needed to permittopotactic polymerization of diacetylene. Topotactic polymerizationrelates to ordering the neighboring polymerizable diacetylenicfunctionalities in a parallel to each other fashion. This ordering isacquired in diacetylene containing chains by cooling the lipid vesiclesbelow their phase transition temperature or T_(m). See Ticke, B., et al,J. Polym. Sci., Polym. Chem. Ed. 7, 1631-1644 (1979) and Lever et al,Biochim. Biophys. Acta 732, 210-218 (1983).

The diacetylenic SUVs do remain stable at temperatures down to about2.4° C., almost 40 degrees below T_(m), where differential scanningcalorimetry shows that a phase transformation occurs. See T. G. Burke,et al., Chem. Phys. Lipids., 48, 215 (1988). Although these smallvesicles do not turn into tubules upon cooling, as discussed above,these SUV diacetylenes fail to polymerize to provide an extendedconjugation due to curvature constraints. Such extended conjugation isdesired to provide darker color which results in better visibility.

Giant unilamellar vesicles (GUVs) produced from diacetylenicphospholipid, 1,2 bis(heptacosa-8,10-diynoyl)-sn-glycero-3-phosphocholine (DC_(6,15)PC) haverecently been prepared which are generally 10 to 100 times larger thantypical vesicles by applying an electric field to the aqueous dispersionmaintained above its chain melting transition temperature (T_(m)) of58.9° C. See Alok Singh, Paul E. Schoen, Marie-Alice Guedeau-Boudeville,Chem. Phys. Lipids., 94, 53-61 (1998). There has been no disclosure ofthese new GUVs being used in colorimetric sensors.

OBJECTS OF THE INVENTION

It is an object of this invention to produce giant unilamellar vesicles(GUVs) with a large radius of curvature which can polymerizeeffectively.

It is a further object of this invention to produce giant unilamellarvesicles with a large radius of curvature which are polymerized andwhich have a polymer membrane that allows the free transport of contentsacross the membrane bilayer so as to eliminate the risk of any colorchange due to osmotic shock.

It is a further object of this invention to produce giant unilamellarvesicles with a large radius of curvature which are polymerized andlarge in size so they may be handled and manipulated individually.

It is a further object of this invention to produce giant unilamellarvesicles with a large radius of curvature which have a size of at least50 microns.

It is a further object of this invention to produce a colorimetricdetector of chemical and biological agents utilizing giant unilamellarvesicles with a large radius of curvature so as to make customizedarrays to enhance detection.

It is a further object of this invention to produce a colorimetricdetector of chemical and biological agents utilizing giant unilamellarvesicles with a large radius of curvature so that individual giantunilamellar vesicles may be probed for color change due to site specificbinding.

These and further objects of the invention will become apparent as thedescription of the invention proceeds.

SUMMARY OF THE INVENTION

A colorimetric detector for chemical and biological agents or toxins ismade of a giant unilamellar vesicle (GUV) having a membrane bilayerwhich is polymerized to stabilize the giant unilamellar vesicle and toprovide extended conjugated polymer backbone, and there is at least oneincorporated molecular recognition site for the chemical and biologicalagents or toxins. The GUV is preferably a polymerizable diacetylenicGUV. In this case, the polymerization to stabilize the giant unilamellarvesicle is done by crosslinking the acyl chains in the membrane bilayerof the diacetylenic GUV. Upon the binding by the chemical and biologicalagents or toxins, the detector exhibits a color change in the visibleregion of the spectrum. Examples of incorporated molecular recognitionsites are di or polysachharides substituted with a long alkyl chainpreferably containing diacetylenic moiety. Preferable examples areN-octadecyl maltobionamide (C-18 maltonamide) and N-octadecyllactobionamide (C-18 lactonamide). The GUV is preferably made from 1,2bis-(alkadiynoyl)-sn-glycero-3-phosphocholine DC_(m,n)PC where m=2-16and n=7-16, and m+n≦20 carbon atoms. A particularly preferred materialis 1,2 bis-(Tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholineDC_(8,9)PC. The GUV in the detector has a large radius of curvature ofat least 50 microns and the size of the GUV is about 10-300 microns. TheGUV can be used on a substrate to detect the presence of specificspecies or it can be used by itself as a substrate to detect thepresence of specific species.

The colorimetric detector is used for the detection of chemical andbiological agents or toxins in a test sample by exposing the test sampleto the colorimetric detector which is made of giant unilamellar vesicles(GUV) having a membrane bilayer which is polymerized to stabilize thegiant unilamellar vesicle and to provide extended conjugated polymerbackbone and which has at least one incorporated molecular recognitionreceptor site for the chemical and biological agents or toxins. As aresult of the interaction of the chemical and biological agents with thereceptor sites a colorimetric detection signal is produced.

The colorimetric detector can be used in a colorimetric detectorapparatus for detecting chemical and biological agents or toxins. Theapparatus has a chamber with a series of passageways for analyzing thesampling fluid containing the colorimetric detector described above.There is a chamber inlet for sampling a fluid containing the chemicaland biological agents or toxins, a means for reading the output of thecolorimetric detector, and a chamber outlet for the sampling fluid. Thedifferent passageways can have different detectors for various agentsand toxins. The passageways can have an inlet filter which canpreferably be a 2-10 micron filter and a similar filter can be used onthe outlet side of the passageway. The passageway can have capillariesfilled with the GUVs. The colorimetric detector apparatus can have awindow for optical read out and a commercial monitor can be used to readthe output of the colorimetric detector. The detector is capable ofhandling samples present in air or in water.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a device for monitoring samples by using thecolorimetric detector.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The GUV has been receiving much attention recently in the literature.They are vesicles with diameters of about 10-300 microns. One reason fotthe attention is because it transforms what has always been amicroscopic object, the vesicle, into something on a macroscopic scaleof up to 2-3 tenths of a millimeter in diameter. With this relativelylarge radius of curvature the GUV still maintains a single bilayer wallthickness. The GUV has become a robust, transportable version of theLangmuir Blodgett (LB) film at the air-water interface.

We have found diacetylenic lipids provide a stable vesicular form over awide temperature range below their phase transition temperature, so itallows experimentation with non-tubular morphology below T_(c). As tonomenclature, when cooling the hot lipid dispersion to room temperatureacyl chains of the lipid transform from melted to crystalline phase.Temperature at which this transition is observed is called T_(c.) It issimilar to the transition temperature when the crystalline chains meltsupon heating the dispersion also called melting transition temperatureT_(m). Previously, all but the smallest vesicles of this lipid wouldtransform into tubules below the phase transition temperature. We havefound that by polymerizing these GUVs they do not collapse at roomtemperature and that the polymerization provides both stability andimproved color.

While investigating GUVs, we have found that GUVs are polymerizable andthat GUVs are porous. We have also found that GUVs can befunctionalized. The preferred diacetylenic GUVs in their polymerizedform are a permanent plastic bubble which resembles a real life soapbubble, they are freeze-dryable, they are a stable platform, and theyare highly porous. The advantage of having a porous vesicle means that achange in the surrounding medium will not give an osmotic shock to thevesicle and therefore disrupt the sensing mechanism. The GUVs provide ahospitable environment for biomolecules, maintaining hydration whichpreserves functions for enzymes, for instance.

We have further found these GUVs by virtue of their size and lack ofstringent curvature, and due to their relatively large radius ofcurvature lead to extensive polymerization and thus produce longerconjugation polymers which are superior for detection. Such a longerunstressed conjugated polymeric backbone produces better signal andbigger changes upon binding of linked ligand sites to pathogens asopposed to small vesicles or polymeric films.

The production of a colorimetric detector based on the GUV is done in aseries of steps. First, a lipid which will form a giant unilamellarvesicle (GUV) is dissolved in a solvent along with a glycosurfactantthat will serve as a molecular recognition site. Next the solvent isremoved to produce a film with the glycosurfactant incorporated in thelipid. Then the film is hydrated and subjected to conditions to form aGUV. A preferred condition is treating the mixture to an alternatingelectric field while maintaining the temperature above the chain meltingtransition temperature of the lipid to produce electrical swelling andformation of the GUV. Then the GUV is polymerized to obtain a stabilizedcolorimetric detector. The resulting colorimetric detector can be testedby demonstrating the specific binding affinity of toxins to surfacebound glycolipids on the polymerized giant vesicles.

To prepare the Giant Unilamellar Vesicles a lipid having the formulaDC_(m,n)PC:

(where m and n represent the number of methylene groups before and afterdiacetylenic moiety in the acyl chain and where m=2-16, n=7-22, andm+n=16-29) was used and it was prepared by following a publishedprocedure. See A. Singh and J. M. Schnur, Synth. Commun., 16, 847 (1986)and A. Singh, L. Lipid Res., 31, 1522 (1990). In this publication thelipid used was a diacetylenic phospholipid 1,2 bis(heptacosa-8,10-diynoyl)-sn-glycero-3-phosphocholine (DC_(6.15)PC)having the chemical structure:

In the nomenclature for this compound D means “di” or two and C refersto “carbon chain” thus DC is a twin carbon chain. The notation m,nrefers to th number of methylenes above (proximal to the head group) andbelow the diacetylene group in the acyl moiety as shown in the formulaabove.

In the following examples unilamellar, spherical giant vesicles fromDC_(6,15)PC, and saturated distearoyl phosphatidylcholine (DSPC) wereprepared by following a reported procedure modified to this application.See M. I. Angelova, D. S. Dimitrov, Mol. Cryst. Liq. Cryst. 152, 89-104(1987).

The preferred diacetylenic GUVs are prepared by treating the lipid to analternating electric field while being heated to produce electricalswelling and formation of the GUVs. The GUVs were cooled below theirT_(c) temperature and polymerized for stability.

Having described the basic aspects of the invention, the followingexamples are given to illustrate specific embodiments thereof.

EXAMPLE 1

This example illustrates a preferred method for producing a GUV. A 10 mlchloroform/methanol (9:1) solution (10 mg/ml) of a lipid having theformula DC_(m,n)PC:

where m and n represent the number of methylene groups before and afterdiacetylenic moiety in the acyl chain was spread on two conductiveIn-SnO₂-coated glass plates (3.5 cm×1 cm) and then dried under highvacuum for approximately 1 hour to obtain a thin lipid film. The plateswere then fixed, face-to-face, about 1 mm apart, by sandwiching a Vitonrubber spacer between them. After applying an 0.5 V, 10 Hz AC voltage(Hewlett Packard 332A Function Generator) the cell was filled with a 50mM sucrose solution and the aperture was sealed with Vitrex.

This sealed cell was placed in an oven at 72° C., during the process ofelectrical swelling. The alternating-current voltage was increasedslowly from 0.5 V to 1.5 V over 1 hour. After 6 hours the frequency wasdecreased to 5 Hz and the swelling was continued for an additional 4hours.

The GUVs thus produced were stored at 72° C. The giant vesicles wereobserved by a reverse-phase microscope (Nikon Diaphot-TMD, objective×20). When the lipid was a diacetylenic phospholipid 1,2bis(heptacosa-8,10-diynoyl)-sn-glycero-3-phosphocholine (DC_(6,15)PC)the average diameter vesicles obtained were >100 μm.

Diacetylenes in bilayers polymerize efficiently below their chainmelting transition temperature T_(m) because at this lower temperatureall of the chains are extended to attain an all-trans conformation,which is needed for polymerization to occur. Smaller multilamellarvesicles (MLVs) usually transform into tubule structures when cooledjust below their phase transition temperature T_(m).

EXAMPLE 2

This example illustrates the stability of the GUV on cooling anddemonstrates GUVs can be cooled down to about 28° C. below their T_(m)without losing their morphology. This permits production of longerconjugation and consequently good color.

GUVs as made in Example 1 from DC_(6,5)PC upon slow-cooling below theirT_(m) did not show any morphology transformation. They remained asbubbles and did not become tubules, smaller vesicles or simply flatsheets. The effect of cooling on the morphology was studied by usingoptical microscopy. The swelling cell, which is the container in whichGUVs are created, produces many giant vesicles with diameters up toabout 300 micrometers, was directly placed in a temperature regulatedsupport connected to a thermostated bath. The temperature changes weremonitored by a microcomputer thermometer. The thermocouple was insertedbetween the cell and the regulated heating support, which providedaccurate readings of the cell temperature. GUVs were examined formorphological changes by lowering the temperature from 70 to 30° C. asit was cooled from 70 to 30° C. at a rate of about 1° C./min.

A series of video images were taken and examined by optical microscopyof a 260 micrometer diameter GUV of the diacetylenic lipid which showedat what temperature the GUV began to rupture and collapse. For, example,six photos taken at the following temperatures: 32.1° C.; 31.9° C.;31.8° C.; 31.6° C.; 31.55° C.; 31.5° C. At 32.1° C. a dark spot appearedat the equator of the spherical vesicle. This could be the weak part ofthe membrane caused by the defect due to mismatch in lipid registeringwith the next molecule. Disintegration of the membrane began at thispoint, spreading rapidly all around the circumference of the vesicle,resulting in a lipid aggregate of unknown morphology. These photos showthat the collapse occurs at a triggering temperature and the collapseoccurs rapidly. These images illustrate the progress of collapse. Thereis only a minute amount of time for the temperature to change during thecollapse.

MLVs of DC_(6,15)PC with diameters near 1 micrometer and LargeUnilamellar Vesicles (LUVs) which are vesicles with diameters equal toor more than 1 micrometer transform into tubules near T_(m) at 59° C.GUVs, on the other hand, with much larger diameters of 200 micrometersor more disintegrate at a temperature 26 degrees cooler than its T_(m).Thus the large radius of curvature of the GUV accompanies a largertemperature window of stability below T_(m) for the vesicular structuresand facilitates efficient polymerization at the same time.

This example illustrates the unique shape of the GUV. Unlike the SUV orthe MLV, the GUV retains its shape upon cooling to far below its T_(m)until it finally collapsed.

EXAMPLE 3

This is a comparative example illustrating the effect of cooling onsaturated 1,2 distearoyl phosphatidylcholine (DSPC).

Saturated 1,2 distearoyl phophatidylcholine (DSPC) having the formula:

was cooled from 60.0° C. to 42.0° C. and rate of cooling was 1° C./min.The liquid to gel phase transition temperature T_(m) occurred at 47.8°C.

This example illustrates the difference between diacetylenic GUVs asevaluated in Example 2 and DSPC GUVs and it illustrates the uniquenessof a GUV derived from a diacetylenic lipid. Saturated DSPC GUVs don'tshrunk in size upon cooling so they are stable, but they have no color.The reason for this is that upon polymerization diacetylene producesdeep blue to red color showing that diacetylenes are polymerized toconjugated polydiacetylene. The DSPC, on the other hand, does notpolymerize because it doesn't have any diacetylene functionality. Thediacetylenic GUVs do not gel like saturated DSPC GUVs and they are notstable down to room temperature such that they will collapse unless theyare polymerized. However, as will be illustrated in the next example,polymerization solves that stability problem.

Polymerization of GUVs of DC_(6,15)PC can be performed by directlyirradiating the sample cell with UV light from a mercury pen lamp.

EXAMPLE 4

This example illustrates that GUVs do not change morphology upon cooling<T_(m), a temperature necessary for polymerizing vesicles and it servesto demonstrate efficient polymerization.

Polymerization of GUVs of DC_(6,15)PC was performed by directlyirradiating a sample cell with UV light from a mercury pen lamp. Thetemperature was maintained at 50° C., well below T_(m) (59° C.), so thelipid would be in its low temperature ordered phase. In this phase theacyl chains will be in the all-trans configuration, with theirdiacetylenic units packed close together, which is the favorablealignment required for topotactic polymerization. As the polymerizationprogressed, the contrast of the GUV in the optical images began todecrease. After about 30 seconds had passed, the contrast had droppedsubstantially, and the GUV had become very difficult to distinguish fromthe surrounding medium. The initial high contrast was because inside thevesicle the contents are different from outside. The subsequent lowercontrast means the GUV has become porous and the inside and outsidecontent are the same.

This example confirms that GUVs polymerize efficiently leaving a highlypermeable membrane which is stable at lower temperature. This highpermeability is suitable for a detection system because unrestrictedtransport of medium across bilayers will not cause any osmotic shock,which could have caused some stress on the polydiacetylene backbone.

The following examples illustrate the molecular recognition involving aninteraction between polymerizable vesicles made from 1,2bis-(Tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine and twomolecular recognition (receptor) sites N-octadecyl lactobionamide (C-18lactonamide) and N-octadecyl maltobionamide (C-18 maltonamide) with twochemical and biological ligands in the form of lectins, concovalin A(ConA) and peanut agglutinin (PNA).

EXAMPLE 5

This example describes how vesicles with surface bound glycolipids wereprepared.

Two glycosurfactants, derived from disachharide maltose and lactose,N-octadecyl maltobionamide (C-18 maltonamide) and N-octadecyllactobionamide (C-18 lactonamide) were incorporated in the polymerizablediacetylenic phospholipid (DC_(8,9)PC) vesicles. The vesicles contained5 mol percent glycosurfactant incorporated in them. Typically, 5.7 mgglycosurfactant and 17.4 mg polymerizable lipid, DC_(8,9)PC, weredissolved in 10 mL (1:1) chloroform:methanol. The solvent was thenremoved in a slow stream of nitrogen gas leaving behind a thin film. Thethin film of lipid was hydrated by adding 10 mL of appropriate buffersolution (Tris HCl, pH 6.4 for C-18 maltobionamide and Phosphate buffer,pH 7.4 for C-18 lactobionamide). Hydration was completed by heating at30° C. for 30 minutes followed by brief sonication to ensure uniformdispersion. Typical concentration of the lipid in dispersion was keptaround 2 mg/mL. After this insertion of the glycolipid into vesiclemembrane the vesicles were then polymerized by exposing to 254 nm light.Any large pieces in the dispersion were removed by centrifuging thevesicle dispersion.

EXAMPLE 6

This example describes specific binding affinity of toxins to surfacebound glycolipids.

The specificity of surface available galactose or glucose functionalityof the two surfactants used to make the two vesicles with surface boundglycolipids in Example 6 was examined by exposing them to two lectinsPNA (galactose specific) and ConA (glucose specific).

For sugar-lectin binding assays, a dilute solution of lectin(concentration was chosen arbitrarily) was prepared in appropriatebuffer. The tris buffer was used for Con A and the phosphate buffer wasused for PNA lectin. Agglutination or binding affinities were measuredby adding 100 ml lectin solution to 400 ml vesicle dispersion and thenrecording the absorption at 400 nm against time until a plateau isobtained.

The results showed for the binding of lectins with sugar head-groupsincorporated in the polymerized vesicles there was a binding affinitybetween C-18 maltonamide and Con A and a binding affinity between C-18lactonamide and PNA.

The polymerized vesicles by themselves do not show any affinity towardseither ConA or PNA (data not shown). The results also showed a negativeaffinity between lactonamide and ConA and a negative affinity betweenmaltonamide and PNA.

The detection scheme described above has been with the sample in anaqueous medium. Using just dry vesicles Will not show a color changeupon binding. However, the vesicles can be stored in freeze-dried formfor a long period and then they can be hydrated before use.

A device which can monitor either samples in the air or in a liquid isillustrated in FIG. 1.

The detector in FIG. 1 has an inlet (12) through which either air orwater can be introduced. The fluid flows into a series of passageways(14) which have an inlet filter 16 which can be a 2-10 micron filter andthen into capillaries (18) filed with GUVs containing receptors specificfor individual toxins. The GUVS are preferably in buffer or water. Thefluid leaves the passageways through an outlet filter (20) which can bethe same type as the inlet filter (16) and finally the fluid exitsthrough the device outlet (22). The device can have a quartz or glasswindow (24) for optical read out and a monitor (26) can be used. Colorchanges can also be monitored by using a portable commercialspectrophotometer.

The current approach involving giant unilamellar vesicles is farsuperior for color detection for the following reasons. The giantunilamellar vesicles with their large radius of curvature willpolymerize well. The polymer membrane of the GUV allows free transportof contents across membrane bilayer, which eliminates the risk of anycolor change due to osmotic shock. The GUVs may be handled andmanipulated individually due to their relatively large size. Thisability to add various receptors to the GUVs permits making customizedarrays to enhance detection. Finally, each individual GUV may be probedfor color change due to its site specific binding.

It is understood that the foregoing detailed description is given merelyby way of illustration and that many variations may be made thereinwithout departing from the spirit of this invention.

What is claimed is:
 1. A method of making a colorimetric detector forchemical and biological agents or toxins comprising: dissolving in asolvent a lipid which will form a giant unilamellar vesicle (GUV) and aglycosurfactant which acts as a molecular recognition site, wherein theglycosurfactant is a di or polysachharide substituted with a long alkylchain containing a diacetylenic moiety; removing the solvent to producea film with the glycosurfactant incorporated in the lipid; hydrating thefilm and forming a GUV; and polymerizing the GUV to obtain acolorimetric detector.
 2. A method according to claim 1, wherein the GUVis a polymerizable diacetylenic GUV.
 3. A method according to claim 1,wherein the GUV is formed by treating the lipid to an alternatingelectric field while maintaining the temperature above the chain meltingtransition temperature of the lipid to produce electrical swelling andformation of the GUV.